Defining the gate domain of the filamentous phage secretin pIV

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Massey University
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Secretins are a family of large outer membrane proteins with large-diameter lumens (5-10 nm). This allows them to transport bulky substrates, including folded proteins, or assembled macromolecular structures – filamentous phages and type IV pili. Many proteins exported by secretins are essential for virulence of Gram-negative pathogens. Such a large channel would ordinarily sensitize the bacterial cell to noxious agents. However, secretins do not - the presence of a mobile septum, or gate, across the lumen of the channel prevents access by noxious agents. Despite the importance of the gate in secretin function, the sequence identity of the gate residues is unknown. In this study, in vivo random mutagenesis was used to map amino acid residues involved in gating the filamentous phage secretin - pIV. This approach has identified 34 residues that are involved in the gating mechanism. These residues are predominantly located within the secretin homology domain and organised into two clusters; GATE1 (39 residues) and GATE2 (14 residues). A number of isolated point mutants sensitised Escherichia coli to bile salts and antibiotics. These findings allowed the construction of a site-directed deletion mutant of GATE2, confirming the gate function. This thesis mapped, for the first time, a secretin gate. Given the success of the mutagenesis approach used in this thesis, the method here will be applicable to secretins of pathogenic bacteria and other outer membrane channels whose gate regions have not been determined as yet. Knowing the gate regions of “pathogenic” secretins in turn will help design secretin-targeting antimicrobials.
Appendix 5 removed due to copyright restrictions. Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P.,...Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular microbiology, 76(1), 133-150. doi:10.1111/j.1365-2958.2010.07085.x
Secretin gates, Secretins of pathogenic bacteria